BB lecture 12-3 recombinant DNA, PCR

BB lecture 12-3 recombinant DNA, PCR - Chapter 20...

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Chapter 20 (pp.384-391) Recombinant DNA, cloning, PCR Learning objectives: Be able to outline the general steps in making recombinant DNA & cloning a gene Describe how DNA clones of interest may be screened and identified Be aware of potential difficulties when using prokaryotes to produce eukaryote proteins Distinguish between a cDNA library and a genomic library Be able to diagram how DNA is amplified by the polymerase chain reaction
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Recall Griffith’s work… Figure 16.2 Can the genetic trait of pathogenicity be transferred between bacteria? Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells. EXPERIMENT RESULTS CONCLUSION Living S (control) cells Living R (control) cells Heat-killed (control) S cells Mixture of heat-killed S cells and living R cells Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample. Term – transformation refers to uptake of extracellular DNA by the cell
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Other methods of introducing DNA into eukaryotic cells: electroporation , applying a brief electrical pulse to create temporary holes in plasma membrane Directly inject DNA into cells using microscopic needles Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination
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Cloning a Eukaryotic Gene in a Bacterial Plasmid In gene cloning, the original plasmid is called a cloning vector bacteriophage are also common cloning vectors A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there
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In recombinant DNA , nucleotide sequences from two different sources are combined in vitro into the same DNA molecule
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Recombinant DNA and cloning – Basic Steps 1. Vector DNA and gene-source DNA are isolated
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Restriction enzymes are used to make recombinant DNA Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites (the cuts are “restricted” to specific sites – depending on base sequence) A restriction enzyme usually makes many cuts, yielding restriction fragments The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary “sticky ends” of other fragments DNA ligase is used to join restriction fragments together
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Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA Restriction site DNA 5 3 5 3 G A A T T C C T T A A G Restriction enzyme cuts the sugar-phosphate backbones at each arrow DNA fragment from another source is added. Base pairing of sticky
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This note was uploaded on 03/19/2008 for the course BIO 311C taught by Professor Satasivian during the Fall '08 term at University of Texas at Austin.

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BB lecture 12-3 recombinant DNA, PCR - Chapter 20...

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